A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on a floppy diskette.
1. Field of the Invention
The present invention is broadly concerned with multiple-peptide channel assemblies which provide transport of anions through epithelial cell membranes wherein the preferred peptides have from about 16-31 amino acid residues and are soluble in water to a level of at least 5 mM; such channel assemblies can be used in the treatment of diseases such as cystic fibrosis (CF) and adult polycystic kidney disease (APKD), as well as in the killing of undesirable cells. More particularly, the invention pertains to such channel assembly forming peptides, and corresponding methods of use, wherein the peptides are derived from a segment of a native (i.e., naturally occurring) channel protein and have their water solubilities enhanced by modification of the C- or N-ends thereof modified with a plurality of polar amino acid residues such as lysine. Still more particularly, the invention pertains to derivatives of the M2GlyR sequence which remain predominantly in monomer form when in solution, have a desired amount of helical configuration, and alter the transepithelial electrical resistance of cells to a greater extent than was heretofore possible.
2. Description of the Prior Art
Introduction.
A major problem in CF is the inability of airway epithelia to secrete fluid. The resulting changes in the composition of the mucous coating the airway epithelia result in infection and subsequent inflammation, scarring, and eventual pulmonary destruction. The basis of the problem is the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane of the epithelial cells. This leads to an increase in the absorption of salt and water and an inability to respond to appropriate stimuli by secreting chloride and water. CFTR is a chloride channel; in addition it down-regulates sodium channels and up-regulates another population of chloride channels, the outwardly rectifying chloride channel (ORCC). These properties of CFTR enable the airway cells to secrete chloride and this drives the secretion of sodium and water.
A synthetic-23-residue xcex1-helical peptide designated M2GlyR forms anion-selective channels in phospholipid bilayers. This peptide has the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding a subunit of the inhibitory glycine receptor.
The origin and properties of M2GlyR.
The glycine receptor is a membrane protein present in post-synaptic membranes. Binding of glycine activates a Clxe2x88x92 conducting channel, leading to hyperpolarization of the membrane and inhibition of the synapse. The receptor consists of two major glyco-polypeptides, an a subunit of 48 kd and a xcex2 subunit of 58 kd, and a receptor-associated cytoplasmic protein of 93 kd. Strychnine, an antagonist of the glycine receptor, binds only to the xcex1 subunit. Messenger RNA corresponding to this subunit leads to the expression of functional, glycine-activated, Clxe2x88x92 channels upon injection into Xenopus oocytes.
The glycine receptor channel in cultures of embryonic mouse spinal cord is selective for monovalent anions, with conductances of 27 and 46 pS in 145 mM Clxe2x88x92 solution. Pharmacological studies suggested the presence of two sequentially occupied anion binding sites in the channel. These sites are considered to be the functional correlates of the positively charged amino acids bordering the M2 segment of the xcex1 subunits. This finding led to the development of the synthetic peptide with the sequence of the M2 segment of the glycine receptor.
Electrical recordings from phospholipid bilayers containing M2GlyR showed single-channel conductances of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes in the millisecond range. Single channel events occurred in 0.5 M N-methyl-D-glucamine HCl but not in sodium gluconate, indicating that the channel is anion selective. A transference number for anions of 0.85 was calculated from reversal potential measurements under a 5-fold KCl concentration gradient.
After insertion into the lipid bilayers the monomeric peptides self-assemble to form conductive oligomers of different amplitudes. To gain control over the aggregate number, four identical M2GlyR peptide units were tethered to a 9-amino acid carrier template to form a four-helix bundle protein. This tetramer, incorporated into lipid bilayers, formed channels of uniform unitary conductance of 25 pS. The 49 pS conductance described above is presumed to be due to the presence of a pentamer.
The tetrameric channel was blocked by the Clxe2x88x92 channel blockers 9-anthracene carboxylic acid (9-AC) and niflumic acid (NFA). It was not blocked by QX-222, an analogue of lidocaine and a blocker of cation-selective channels. Strychnine, an antagonist of the glycine receptor, does not block the channel-forming tetramer. Strychnine is presumed to bind to the ligand-binding domain of the receptor exposed to the extracellular surface but not to the channel domain.
Structure of channel forming peptides.
While great strides have been made in the area of channel function and regulation, using the intact protein or in some cases purified channel proteins reconstituted into model membranes, many aspects of channel function remain unresolved. The K+ from streptomyces lividans was crystallized and the structure determined at 3.2 Angstroms. This structure has served as a model for other related channels using homology modeling methodologies. This structure however is for a 4 subunit channel as opposed the five subunit channel proposed for the glycine receptor.
Considerable structural data exists for the related class of channel forming peptides (CFPs). These channels are much smaller in size and contain only a ring of short peptide chains organized around the central ion conducting pore in the lipid bilayer. These channels are unique in that they assemble by the oligomerization of a single peptide. These structures are models for studying the structure and function of the various regulated channels that occur in nature. This class of CFPs includes: the xcex1-aminoisobutyric acid-containing channels such as alamethicin and zervamicin, and a number of toxins and venoms such as melittin, cecropins, mast cell degranulating peptides, and the defensins. Melittin is somewhat of a special case because it forms channels only at low concentrations; at higher concentrations it acts as a lytic agent. In some cases CFPs assemble spontaneously upon insertion into the bilayer while in the remaining cases the assembly requires an electrical potential across the membrane (Vm).
The structure of the channels arising from the assembly of these peptides vary from trimers to hexadecamers associated in the form of helical bundles or xcex2-barrels. The most widely accepted model which is in accord with the model for channel proteins has the helices arranged with their dipoles all pointing in the same direction (parallel). Since CFP channels, unlike authentic channel proteins, are not generated from the association of large protein subunits, alternative stabilization schemes must be invoked to account for the presence of this higher energy arrangement of parallel segments. These could include aligning the dipoles in response to the presence of the membrane potential and/or an increase in the favorable inter-molecular interactions promoted by the parallel assembly. Most CFPs form multiple size bundles of parallel segments (e.g., n=4, 5, 6) that can spontaneously increase or decrease in size upon the addition or deletion of a peptide monomer from the channel assembly. These observations imply that enough information is contained in a single channel forming polypeptide to drive the correct folding, assembly, and activity of these channels.
The activity of these assembled molecules, the opening and closing of the channels on the millisecond time scale, has been ascribed to numerous effects. Three different helical motions have been implicated: the bending and twisting of the helices, rigid-body fluctuations of the entire assembled structure with the lipid bilayer, and rotational motions of the polypeptide around its helical axis. Another hypothesis suggests that channel activity is a consequence of a conformational change that is transmitted along the helical axis. Others suggest that the movement of individual amino acid side-chains could provide this function, and one group contends that an electron transfer could disrupt a hydrogen bonding of four tyrosines in K+ channels.
Fluorescence, Fourier transform infrared spectroscopy (FTIR), and circular dichroism (CD) measured in organic solvents, phospholipid micelles, liposomes, or oriented phospholipid bilayers, have been successfully used to probe the solution and membrane-bound conformations of these peptides. Computer modeling studies have been performed to estimate the energetics of moving a charged ion across a lipid bilayer through a pore generated by a bundle of transmembrane helices. Structural experiments using NMR are yielding important results. In general, these studies have provided several conclusions concerning the solution behavior and membrane interactions of CFPs. Amphipathic helical peptides can exist as monomers and aggregates in solution. Monomers are able to interact much more readily with lipid bilayers and micelles. Depending on the peptide to lipid ratios, type of lipid, ionic strength, pH of the solution, and the hydration of the lipid, the peptide will preferentially orient itself either parallel to or perpendicular to the plane of the bilayer. Many CFPs do not require a potential difference across the bilayer to insert spontaneously into the bilayer. Once in the membrane, the helices associate in a time and concentration dependent manner to form the multistate helical bundles. It is these assemblies that conduct the ions across the bilayer. These studies, when considered together, reveal the transmembrane amphipathic helix to be a dynamic structure. The ability to oligomerize in the membrane into stable ring structures, with a central aqueous pore capable of opening and closing, appears to be driven by the asymmetric alignment of hydrophilic and hydrophobic amino acid residues that seem to obey a unique set of rules.
Putative channel forming segments from large channel proteins behave much like the small naturally occurring CFPs described above. They spontaneously insert into bilayers and self-assemble into an ion-conducting structure, presumably comprised of a parallel array of xcex1-helices. These structures also retain biological activities reminiscent of the native proteins they were modeled after. These structures are reasonable models for exploring both the oligomerization of transmembrane segments and for defining the molecular events that give rise to channel activity. The beauty of this system emanates from the appearance of a measurable activity that arises from the assembly of an amphipathic transmembrane helix. The activity allows measurement of the effects of amino acid substitutions on either the size of the assemblies or the contribution of the residues to ion selectivity or translocation. The number of helices per channel can be precisely controlled, thus preventing multiple oligomerization states, by tethering the helical segments to a peptide backbone during synthesis. The small size of these assemblies makes them ideally suited for NMR structural studies using either detergent micelle solution NMR or oriented bilayer solid-state NMR.
Pharmacological studies have been a relatively recent addition to the single channel analysis of these model CFP channels. Using a four helix bundle CFP derived from the human L-type dihydropyridine sensitive Ca2+ channel, the binding of a local anaesthetic as well as a number of calcium channel blockers with binding affinities on the order of those observed for the full length calcium channel protein have been observed. This avenue of investigation adds a sensitive method of discriminating between channels that truly mimic their parent structures as opposed to those that might produce non-discriminating ionic pores. Once the three dimensional structure for one of the synthetic channels has been solved, rational drug design of both channel agonists and antagonists may be attempted using these coordinates.
Membrane proteins are generally acknowledged to be the most difficult class of proteins for detailed structural analysis. The studies presented above clearly demonstrate the utility of working with channel forming peptides, as model systems, to study events involved in peptide association with the bilayer, insertion into membranes, and assembly into oligomers. The amphipathic helix is a suitable structural motif for the pore of channel proteins that also contributes to the organization, size, function, and stabilization of ionic channels. As an assembled structure these helical bundles can be used to investigate the structure, organization, and function of channels.
Application of synthetic peptides to biological membranes.
A synthetic peptide with the sequence of the M2xcex4 segment of the nicotinic acetylcholine receptor from Torpedo californica forms ion channels in lipid bilayers that emulate those of authentic acetylcholine receptor ion channels. Human erthyrocytes exposed to the synthetic peptide released hemoglobin and K+. Evidently the peptide molecules self-assembled in the membrane to form trimers and pentamers. Extensive evidence indicates that Clxe2x88x92 secretion drives fluid secretion in Madin-Darby canine kidney (MDCK) cells and in cells cultured from the cystic epithelium of the kidneys of patients with autosomal dominant polycystic kidney disease (APKD), and that a Clxe2x88x92 channel is involved in fluid secretion. Indeed there is now extensive data indicating that CFTR is the channel involved in that secretion by APKD cells. Apparently, a net secretion of Clxe2x88x92 into the lumen of the cysts leads to an increase in water volume in the cysts, ultimately resulting in kidney dysfunction. However, although there is a precedent for the application of synthetic channel-forming peptides to cells, no one previously has used channel-forming peptides to treat symptoms of any disease.
U.S. Pat. No. 5,543,399 describes the purification and lipid reconstitution of CFTR protein and CF therapy making use of that protein. There is no teaching or suggestion in this reference of the use of relatively small, easily prepared pure peptides, and particularly peptides which are fragments of channel-forming proteins.
U.S. Pat. No. 5,368,712 teaches the use of small peptides reconstituted in artificial membranes as diagnostic tools. This patent does not describe any therapeutic applications using such peptides.
U.S. Pat. No. 6,077,826, the content of which is hereby incorporated by reference, describes the use of multiple-peptide channel assemblies which transport anions through epithelial cells, synthetic peptides capable of forming such assemblies, channel assemblies which alter the flux of water across these cells, and channel assemblies which alter the transepithelial electrical resistance of cells. These assemblies were based on the M2GlyR sequence and were modified to increase their solubility. However, the activity of these assemblies is limited to about 15 xcexcA/cm2 at a concentration of about 500 xcexcM. Additionally, the peptides of this invention form multimers in solution which have decreased affinity for membranes and suffer from solution aggregation.
Accordingly, what is needed in the art are channel assemblies which exhibit a more potent effect on the transepithelial electrical resistance of cells and transport anions through cells with a greater efficiency. Such peptides should also exhibit greater stability and a lower occurrence of multimers when added to solution.
The present invention solves the problems inherent in the prior art and provides a distinct advance in the state of the art. Peptides of the present invention exhibit an improvement in activity that is about 5 fold greater with respect to activity levels and/or a 10 fold increase in effective concentration than was heretofore possible. The present invention is directed to improved 1) multiple peptide channel assemblies for transport of anions (e.g., Clxe2x88x92) through epithelial cells, 2) synthetic peptides capable of forming such channel assemblies, 3) methods of using the channel assemblies in therapeutic contexts for altering the flux of water across epithelial cells, and 4) multiple peptide channel assemblies which alter the transepithelial electrical resistance of cells. The peptides of present invention exhibit greater stability and reduced solution aggregation which lead to increased bio-availability of the peptides, thereby reducing the amount of peptide necessary to affect a desired response. Additionally, the present invention is directed to peptide sequences which can form channel assemblies having unique cell-killing attributes and which may be useful in combating growth of undesirable cells (e.g. cancer cells).
In preferred forms, the channel assemblies of the invention comprise multiple peptides each having from about 16-31 amino acid residues, and more preferably from about 22-27 residues. The peptides are characterized by the ability of providing, in an embedded channel assembly, transport of anions through a membrane of an epithelial cell and modulation (alteration) of the flux of water through the cell. The peptides are also characterized by their effect on the transepithelial electrical resistance of cell monolayers. Preferred peptides of the present invention will have activity profiles of greater than about 15.0 xcexcA/cm2 in MDCK cells when applied to the MDCK cells at a concentration of about 500 xcexcM. More preferably, peptides of the present invention will have activity profiles of greater than about 15.0 xcexcA/cm2 in MDCK cells when applied to the MDCK cells at a concentration of about 300 xcexcM, and still more preferably at a concentration of about 200 xcexcM, and most preferably at a concnetration of less than about 100 xcexcM. Moreover, the preferred peptides are soluble in water to a level of at least about 5 mM, and more preferably at least about 10 mM, and still more preferably at least about 15 mM. The peptides of the invention also should exhibit at least about 50% helical content (advantageously at least about 65% helical content, and still more preferably at least about 75%) when dispersed in a 20% trifluoroethanol/80% water solution and measured using circular dichroism spectroscopy (CD). Preferred peptides of the present invention are also characterized by greater stability and fewer multimeric forms in solutions. Preferably, the peptides will predominantly form only monomers when dissolved in solution, with just a trace of dimer present. Monomers are preferred due to their higher binding affinity to the membrane. This increased affinity is due to the non-aggregation of the hydrophobic portions which are required for membrane binding, and are therefore available for binding. This increases the overall bio-availability of sequences comprising mainly monomers. When sequences include multimeric forms, the hydrophobic portions aggregate, thereby rendering them unavailable for binding and decreasing their bio-availability. For peptide sequences having cell-killing attributes, such sequences will induce a negative effect on the resistivity of cell monolayers, eventually leading to a breakdown of the monolayers and the death of the cell. This negative effect is thought to break down the junctions between cell layers. However, this effect is not seen when isolated cells are exposed to these peptide sequences.
In the case of CF therapies, the channel assemblies are embedded in the cytoplasmic membrane of affected epithelial cells. These peptides spontaneously insert into the cytoplasmic membrane on contact, and spontaneously aggregate within the membrane to form a channel assembly having a hydrophilic internal pore through which Clxe2x88x92 may pass, and an lipophilic external surface allowing solubility of the assembly in the membrane. Preferably, the peptides making up the channel assemblies are identical. In another use, the peptides may spontaneously insert into the basolateral membrane of renal epithelial cells in order to inhibit the flux of water into the adjacent cysts.
The peptides ideally have an amino acid sequence based upon the sequence of the M2GlyR peptide which has been subsequently modified by the addition of multiple polar amino acid residues on the C- or N-ends. C-K4-M2GlyR (PARVGLGITTVL-TMTTQSSGSRAKKKK)(SEQ ID No. 2), was initially chosen as the lead CF drug compound due to its higher solubility in water, higher proportion of monomer in solution, and its ability to better mimic the pharmacology associated with the unmodified M2GlyR sequence. The second peptide N-K4-M2GlyR (SEQ ID No. 3) (KKKKPARVGLGITTVLTM-TTQSSGSRA), upon closer analysis, shows an approximately 50% higher level of conductance than the C-K4 peptide. It also appeared to form channels faster and had channels with improved stability. This increase in activity may be due to a structural difference that was been observed in modeling studies. In addition to these differences, other disparate properties such as degrees of aggregation in solution, rates of aggregation in physiological buffers and sensitivities to different channel blocking agents have been noted between the two peptides. These artificial anion conducting channels appear to be regulated by potassium channels located in the baso-lateral membrane. The anion conductance seen with C-K4-M2GlyR, is most likely, the result of a novel chloride conductance pathway. These measurements were obtained using Madin-Darby canine kidney cells, the human colonic epithelial cell line (T84), and airway epithelial cells derived from a human cystic fibrosis patient (IB3-1). N-K4-M2GlyR also acts to form a novel chloride conductance pathway but yields an approximately 50% increase in short circuit current (Isc) over that of C-K4-M2GlyR as described above. This increase in activity may be due to a structural difference that has been observed in modeling studies. In recent studies, both peptides were shown to restore glutathione transport in cultured CF monolayers. Again, C-K4-M2GlyR was active but to a much lesser extent, thereby reaffirming the theory that N-K4-M2GlyR functions better than C-K4-M2GlyR. The fact that N-K4-M2GlyR can be regulated by the cell through baso-lateral K+ channels and that its presence in compromised CF cell line helps restore glutathione transport, suggests that this peptide improves the health of CF cells.
However, one of the obstacles to generating better channel forming sequences based on the M2GlyR sequence has been the multi-state nature of N-K4M2GlyR and C-K4M2GlyR in solution. Therefore, in an attempt to reduce the amount of solution aggregation, a new family of peptides based on the M2GlyR sequence was created using a modular approach. The modules consist of the 11 amino acid residue segments surround the central leucine (L) residue: module A=PARVGLGITTV (SEQ ID No. 48) and module B=TMTTQSSGSRA (SEQ ID No. 49). Using this nomenclature, the native sequence for M2GlyR is A.L.B. Derivative sequences were created using module A (PARVGLGITTV), module B (TMTTQSSGSRA), the A module in reverse (VTTIGLGVRAP) (SEQ ID No. 50), referred to as a, the B module in reverse (ARSGSSQTTMT) (SEQ ID No. 51) referred to as b, Axe2x80x2 (AARVGLGITTV) (SEQ ID No. 52) having an alanine substituted for the initial proline, and axe2x80x2 (VTTIGLGVRAA) (SEQ ID No. 53) which is the Axe2x80x2 module in reverse. New sequences were generated by combining the six modules, A, B, a, b, Axe2x80x2, and axe2x80x2, in all possible combinations separated by the leucine normally found between these modules in the wild-type sequence. Sequences such as A.L.A, a.L.a, a.L.A, Axe2x80x2.L.b, etc. were synthesized. In other sequences comprising the six modules, tryptophan (W) was used between the modules, as opposed to the naturally occurring leucine.
Preliminary results indicated those newly designed peptides with a propensity to form an alpha-helical structure (assessed by CD in 20% trifluoroethanol (TFE) in H2O), were more likely to promote anion secretion across epithelial cell monolayers. For peptides which have an activity less than 1, such peptides generally have less than 20% helicity and exhibit a structure more closely related to a beta structure, which has difficulty forming pores in membranes. For peptides having activities greater than 1, such peptides generally have greater than 20% helicity which helps form the bundle and thereby, form a pore through a membrane.
Based upon success in solubilizing transmembrane sequences, amino-terminal lysyl adducts were added to the C- and N-termini of the new modular mutants. C- and N-K4-(A.L.a) (PARVGLGITTV-L-VTTIGLGVRAP) exhibited higher activity than had previously been found in the prior art. Because this sequence is a palindrome, the amino- and carboxyl-terminal lysyl adducts allow for testing the effects of the helical dipole on anion transport. Both adducts have shown increased Isc in MDCK epithelial cell monolayers with half maximal effects observed at or below 30 xcexcM, a nearly 10-fold improvement over any peptide previously characterized in the C- and N-K4 M2GlyR family. C-K4 A.l.a, however, produced channels that were toxic to the cell while N-K4 A.l.a produced equally high conductance levels (up to 45 xcexcAmp/cm2) that were not harmful to isolated cells. SDS-PAGE gels of cross-link ed peptide revealed that the N-K4 A.l.a is greater than 90% monomeric with only a trace of dimer and nothing higher.
Computer modeling studies were subsequently performed on many of the known active sequence-using conditions that mimicked folding in the membrane phase (low dielectric). Under these conditions an unexpected result was obtained. The structure for C-K4 M2GlyR as well as the palindrome C-K4 A.L.a had the four lysine residues folded back at the C-terminus. Hydrogen bonds were formed between two of these lysine residues and the helix backbone. In contrast, the lysine residues at the N-terminus of the palindromic sequence C-K4 A.L.a extended away from the helix and were not H-bonded. These preliminary results were consistent with the results obtained from the C-capping of a synthetic peptide modified with a single lysine at the C-terminus determined from NMR. The C-capped structures also showed a moderate compression in the second turn of the helix at the amino terminus. The implications of this structure on function are significant for transmembrane sequences. In designing the water soluble N-K4 and C-K4 derivatives, it was assumed that the lysine residues would be solvent exposed and also serve to restrict the membrane insertion of the peptides to only one orientation with the lysines remaining outside the membrane. Having the lysines at either terminus should have allowed for the insertion of the peptide with its helix dipole oriented exclusively in one direction. Therefore any assemblage of the inserted sequences should be the result of bundles of parallel helices.
However based on the computer models, the predicted folding back of the lysines in the case of C-K4 M2GlyR suggested that both orientations of the peptide were possible. Most models of the assembled pores formed by channel forming peptides have all helical dipoles parallel. In the case of C-K4 M2GlyR having both orientations of the dipole possible within the membrane would interfere with the assembly of an active synthetic channel. Early modeling studies on M2 have suggested that anti-parallel packing of the helices leads to an assembly without a central pore. Thus, it is likely that an anti-parallel bundle of C-K4 M2GlyR peptides would be non-functional. Before the possibility of multiple orientations within the membrane for C-K4 was recognized, the working hypothesis was that the higher the concentration of monomer (in solution), gave rise to higher activity in cells. Now it appears that one must also consider (in the case of C-K4 peptides) the concentration of peptide in the membrane with the correct orientation of dipoles as well as the competitive inhibition that might arise from complexation of helices with the opposite dipole.
Physical data from other experiments support the modeling data described above. In a set of cross-linking experiments designed to characterize aggregates of the two sequences in water, N-K4 M2GlyR gave a ladder of bands starting from monomer up to assemblies approaching 36 kDa. However, C-K4 M2GlyR showed only trace amounts of aggregates higher than trimer. Assuming that the lysines are participating in hydrogen bonds with the backbone carbonyls, two postulates can be proposed; 1) the lysine xcex5-amino groups are not readily available for cross-linking or 2) the lysine C-capping disrupts the ability to form the pores in membranes or form aggregates in solution.
A series of single and multi-dimensional NMR experiments were performed on the modular mutants N- and C-K4 A.L.a. Preliminary NMR data on N-K4 A.L.a and CK4-A.L.a shows the fingerprint region (NH to Ca and side chain proton connectivity) of 1H-1H 2D-TOCSY NMR spectra of these peptides recorded in water containing 30% deuterated TFE at 30xc2x0 C. These spectra displayed reasonably sharp lines and the chemical shift dispersion. The upfield shifting of lysine backbone amide protons and down field shifting of side chain NH cross peaks in TOCSY spectra of C-K4-A.L.a in comparison to N-K4-A.L.a clearly indicate that in the CK4 variant, the lysine backbone amine hydrogen might be hydrogen-bonded and side chains folded whereas in NK4 variants, the lysine residues are in extended conformation.
It has also been demonstrated that NMR is a very sensitive technique for assessing the degree of aggregation for soluble peptides based on the M2 transmembrane segment of the brain glycine receptor (M2GlyR), thereby allowing the formulation of the hypothesis that an increase in monomers leads to higher activity. These new initial results indicate that proposed transmembrane peptides have conformational and topographical properties which are observable by NMR spectroscopy and this information confirms the current computer models.
Initially, the most active variant form of M2 was SEQ ID No. 18. Several modifications were made to this sequence and subsequently tested. Some of these variants have enhanced activity in comparison to SEQ ID No. 18. This enhanced activity is present despite the fact that variants of M2GlyR tested included palindromic sequences, mutated sequences, deleted sequences, and combinations of all of these. Some sequences included replacements for one or both proline residues as well as deletion or additions of the central leucine residue(s). Removal of the prolines improves the ease of synthesis and the deletion or addition of leucines has the effect of changing the registry of the lower C-terminal portion of the helix. By removing the central leucine, the lower cylinder of the helix is rotated xe2x88x92100xc2x0. The addition of two three, and four leucines have the effect of rotating the helix +100xc2x0, +200xc2x0 and +300xc2x0, respectively. These changes are required to see if helical packing within the assembly bundles can be altered and make a better behaved pore structure. It is presumed that these sequences having additional leucines will also exhibit an effect on transepithelial electrical resistance of cells.
In another approach, a series of deletion peptides were prepared for both N-K4 M2GlyR and C-K4 M2GlyR. In each case, amino acid residues were deleted from the end opposite the lysine tail. These peptides were designed to test the lengths of the peptides, both N- and C-K4 M2GlyR, that would sustain bio-activity. Additionally, peptides that had amino acid residues deleted from the end opposite the oligo-lysyl tail were prepared and tested.
It is quite apparent from the activity profiles for these sequences that the N-K4 series retains high activity (xcexcA/cm2) over a larger range of peptide lengths than do the C-K4 sequences. Knowing the minimal length sequence that retains full activity could save resources in both the synthesis and subsequent purification of the active sequence. The N-K4 series dropped significantly after 5 residues were deleted. The C-K4 truncated peptides began to lose significant activity with the first deletion. However, activity in many of these truncated peptides remained higher than that determined for either N- or C-K4-M2GlyR. Based on this model, N-K4 p25 and p22 maintain full activity by recruiting the extended lysyl terminus. For these shorter species, the long hydrophobic butyl side chains of lysine allow the xcex5-amino groups to remain within the charged phospholipid headgroup region of the bilayer as the entire helix retains its ability to fully span the bilayer by being pulled down into the membrane. In the case of the C-K4 truncated peptides the lysines are unavailable for this function due to their folded-back conformation and therefore, the peptides begin to lose their ability to fully span the membrane after truncation.
One somewhat unrelated peptide was also generated and tested. SEQ ID No. 47 represents a double ended version of the M2GlyR peptide in that four lysine residues have been added to both ends of the peptide. It was postulated that this sequence would also work since the lysine residues located at the C-terminus are involved in hydrogen bonds. As described above, this C-capping phenomenon should reduce the net charge of the C-terminus and allow it to enter into the bilayer and cross to the other side. Experimental evidence suggests that the peptide induces only about 5 xcexcA/cm2 acitivity but upon protease cleavage yields about 20 xcexcA/cm2.
The present invention also includes a method of altering the flux of water from an epithelial cell presenting first and second spaced apart surfaces. The method broadly includes providing multiple peptides capable of forming a channel assembly with each of such peptides having from about 16-31 amino acid residues therein. These peptides are contacted with the first surface of an epithelial cell thereby causing the peptides to embed therein and alter the flux of water across the cell. In accordance with the method aspects of the invention, the epithelial cells may be selected from the group consisting of CF-affected epithelial cells, e.g., cells selected from the group consisting of airway, intestinal, pancreatic duct and epidymus epithelial cells. In the case of airway epithelial cells, the method further comprises a delivery step immediately preceding the contacting step, wherein the channel-forming peptides are aerosolized inhaled. In another representative method, the epithelial cells are cystic epithelium of an APKD-affected individual, and the first surface of the epithelial cells is the basolateral membrane of such cells.
In another method of the present invention, the resistivity of cell layers can be decreased by contacting the cell layer with a peptide. Preferably, the peptide is a derivative of SEQ ID No. 1 and includes a portion which is palindromic to a portion of SEQ ID No. 1 or to itself. Preferably, this palindromic portion comprises at least about 7 amino acid residues, more preferably at least about 9 amino acid residues and still more preferably, at least about 11 amino acid residues. In order to increase the solubility of these peptides, the C- and/or N-terminuses thereof can be modified to contain a plurality of polar amino acids thereon. A particularly preferred polar amino acid is lysine. The concentration of the peptide necessary for decreasing the cell layer resistivity is preferably up to about 500 xcexcM, more preferably up to about 300 xcexcM, still more preferably up to about 200 xcexcM, and most preferably, less than about 100 xcexcM. Particularly preferred peptides will have at least about 35% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 4-47. More preferably, these peptides will have at least about 50% sequence homology (and most preferably at least about 65% sequence homology) with a peptide selected from the group consisting of SEQ ID Nos. 4-47.
The channel-forming peptides of the invention are normally in the L-stereoconfiguration. However, the invention is not so limited and indeed D-stereoconfiguration peptides can also be used. The latter type of peptides may also have significant advantages as they are not degraded in vivo by proteolytic enzymes nor do they elicit an immune response.
As used herein, the following definitions will apply: xe2x80x9cSequence Identityxe2x80x9d as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are xe2x80x9cidenticalxe2x80x9d at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol.,215:403-410(1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% xe2x80x9csequence identityxe2x80x9d to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 95% identity relative to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5xe2x80x2 or 3xe2x80x2 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.
Similarly, xe2x80x9csequence homologyxe2x80x9d, as used herein, also refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above, and gaps are introduced if necessary. However, in contrast to xe2x80x9csequence identityxe2x80x9d, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence.
A xe2x80x9cconservative substitutionxe2x80x9d refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.
xe2x80x9cIsolatedxe2x80x9d means altered xe2x80x9cby the hand of manxe2x80x9d from its natural state., i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not xe2x80x9cisolated,xe2x80x9d but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is xe2x80x9cisolatedxe2x80x9d, as the term is employed herein.
As used herein, xe2x80x9cderivativexe2x80x9d with respect to M2GlyR, refers to mutants produced by amino acid addition, deletion, replacement, and/or modification; mutants produced by recombinant and/or DNA shuffling; and salts, solvates, and other chemically modified forms of the sequence which retain the activity of the related sequence. Derivatives also include palindromes and reversals of the M2GlyR sequence, palindromes and reversals of portions of the M2GlyR sequence (such as some of the modules generated) and combinations of any of the above.
Sequences having or including a portion having at least about 35% sequence homology with any one of SEQ ID Nos. 4-47 are embraced within the present invention. Preferably, such sequences will have at least about 50% sequence homology with any one of SEQ ID Nos. 4-47, and still more preferably at least about 65% sequence homology with any one of SEQ ID Nos. 4-47.
Additionally, derivatives of the M2GlyR sequence which have their solubilities modified to a level of at least 5 mM and which exhibit similar properties to any one of SEQ ID Nos. 4, 9, 10, 13, 14, 18, 19, 21, 26-28, 32-35, that is sequences which exhibit greater than 15.0 xcexcA/cm2 at a peptide concentration of 500 xcexcM are embraced within the present invention. Preferably, these derivatives will have their solubilities modified by the addition of multiple polar amino acid residues on the C- or N-ends thereof. Moreover, it is preferred that these derivatives exhibit an activity profile of at least about 15.0 xcexcA/cm2 in MDCK cells at a level of 500 xcexcM. More preferably, these derivatives will have an activity profile of at least about 15.0 xcexcA/cm2 in MDCK cells at a level of 300 xcexcM, and still more preferably an activity profile of at least about 15.0 xcexcA/cm2 in MDCK cells at a level of 200 xcexcM. Most preferably, such derivatives will have an activity profile of at least about 15.0 xcexcA/cm2 in MDCK cells at a level of 100 xcexcM. Notably and advantageously, many of the generated sequences exhibited higher activity at lower concentrations than the previously known sequences (SEQ ID Nos. 1-3), thereby allowing a lower concentration of peptide to be used yet resulting in higher activity. It was also observed that, after certain peptide concentration levels had been reached, little or no increase in activity resulted. This tapering off of activity at higher concentrations should permit sequences having high activity at low concentrations to be used with a minimum amount of side effects due to excess peptide being used. Advantageously, this should also result in lower cost per dose, when used in treatment or therapy.